JPS62187417A - Crossreactive and crossdefensive monoclonal antibody againstpseudomonas aeruginosa serum - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention This invention relates to the application of immunological techniques to provide novel materials useful in determining or treating bacterial infections, and more particularly to the application of immunological techniques to Pseudomonas aeruginosa. (Pamudomosaa agr.
The present invention relates to the production and application of human monoclonal antibodies capable of recognizing various serotypes of S. sginoza.

BACKGROUND OF THE INVENTION Durham-negative disease and its most serious complications, e.g.
Bacteremia and endotoxosis are causes of considerable morbidity and mortality in human patients. This is particularly true for the Gram-negative bacterium Pseudomonas aeruginosa, which has been on the rise over the past 50 years with bacterial infections, particularly nosocomial infections.

During the past few decades, antibiotics have become the treatment of choice for the control of Durham-negative disease. However, the continuous high morbidity and mortality associated with Durham-negative bacterial disease indicate the limitations of antibiotic treatment, especially for Pseudomonas aeruginosa (e.g., Antriol, v.a., "Pseudomonas aeruginosa"). : Can antibiotic treatment improve survival?”, J.
, Lab.

Cli%, Mmd, (1978) 94:196-199
reference).

This prompted the search for alternative prevention and treatment methods.

One method considered is strengthening the host's immune system by active or passive immunization. For example, immunization of humans or laboratory animals with whole-cell bacterial vaccines or purified bacterial endotoxins from Pseudomonas aeruginosa is directed against repetitive oligosaccharides of the ribopolysaccharide (LPS) molecule located on the outer cell membrane of Pseudomonas aeruginosa. It has been observed that this results in the development of specific onson antibodies that are primarily directed against the determinants of (Borak, M., Immunoglobulin: Nleyson, J.S., eds., 73-7).
9, U.S. Department of Health and Human Services, 1979).

The aforementioned antibodies have been tested in various animal models (borac,
(above) and in some preparatory food for humans (Young,
L, S., and Borak, M., Pseudomonas aeruginosa, Savers, L1.
ed., pp. 119-132, Hans Huber, 1980
) has been shown to be protective against the lethal effects of Pseudomonas aeruginosa. Furthermore, particularly relevant to the role of these antibodies in humans is the finding of a relationship between survival and high acute serum titers of antibodies against LPS molecules of the infecting strain in patients with Pseudomonas aeruginosa (Borac, et al. M., and Young, L., S., J., Cli%, I%wvsL, 63
:276-286 (1979)).

The above report indicates that immunotherapeutic approaches, such as by administering pooled human immunoglobulins containing antibodies against the infecting strain, could be used to prevent and treat bacterial disease caused by Pseudomonas aeruginosa. It suggests that it is possible.

Human immunoglobulin is defined herein as the portion of fractionated human serum that is enriched with antibodies, in which specific antibodies against strains of Pseudomonas aeruginosa are expressed. This approach to the treatment of diseases caused by Pseudomonas aeruginosa is still under development due to some inherent limitations in using human immunoglobulin components (e.g., Collin 2, M, S, and Roby, R, E: ,, Am.

J, Mad, 76 (3A): 168-174 (
(1984)). Commercially available products utilizing these ingredients are not currently available.

One of the aforementioned limitations with immunoglobulin components is that they consist of a pool of samples from one or more sources, and such samples cannot be predicted for the presence of a particular anti- P. aeruginosa antibody. It is selected. This pooling results in an averaging of the individual antibody titers so that there is only a modest increase in the resulting titer of the desired antibody.

Another limitation is that the preselection process itself requires expensive continuous screening to ensure product consistency. Despite these efforts, immunoglobulin products still have considerable variability from punch to batch and between products from different regions.

Another limitation inherent in immunoglobulin compositions is that their use results in large amounts of proteinaceous substances (such as those associated with recently acquired immunodeficiency syndrome, or AIDS IfC) that have the potential to cause undesirable biological effects. (including viruses, such as those shown to be associated with the virus). The combination of low titers of desired antibodies and high content of foreign substances often limits the amount of specific and thus beneficial immunoglobulin that can be administered to a patient to suboptimal levels.

In 1975, Uhler and Milstein reported that certain mouse cell lines could be fused with murine pancreatic cells to create hybridomas, each secreting antibodies of a single specificity, i.e., monoclonal antibodies. did(
Uhler, G., & Milstein, C., Natur.
E, 256:495-497 (1975)). With the advent of this technology, it became possible to produce highly specific murine antibodies directed against specific determinants on the antigen. However, the mouse monoclonal antibody or the composition of the antibody may be difficult to use, especially when the mouse monoclonal antibody is used in tests for the treatment of certain human diseases.
Antibodies have important drawbacks for use in humans, in light of the observation that they have been observed to produce an ineffective immune response (Levy, R.L. and Miller, R.A. ,As%,Rav,Ahd,,34:10
7-116 (1983)).

Using hybridomas, Sadoff et al. report the production of a mouse monoclonal antibody of 11M5 directed against the O side chain determinant on the LPS molecule of a specific serotype of Pseudomonas aeruginosa ( 1982 1ntaraeianca Co
%farancg onAntimtcrobial
Agents and Chamot rapy abstracts). They further reported that this murine antibody protected mice against a lethal challenge with P. aeruginosa of the same serotype (ie, a homologous serotype) to which the antibody was directed. Several later papers have detailed the development of murine and human anti-Pseudomonas aeruginosa LPS monoclonal antibodies of varying specificity; for example: Sawada, S., et al., J., Igf.
Dis,.

150:570-576 (1984); Sadoff, J. et al., Antibiot-Chgmother, 36:
134-146 (1985); Hankotsuk, R6 et al.
I? lfg c t , 11rLmun.

37:166-171 (1982); Sidak, A.W.
.

Ronin Antibodies, edited by Engelman, E.G., et al., 16.
7-185Jj, Blenum Publishing Corporation (1985) and Sawada, S., et al., J. hsf.
,Dig,.

152:965-970 (1985). Anti-Pseudomonas LPS
Pending U.S. Patent Application Nos. 734,624 and 828 for Sero-Serotype Specific Human Vaccine Antibodies and Immunotherapeutic Applications
．． QQ5, which are incorporated herein by reference.

There are several advantages to utilizing monoclonal antibodies specific for a single serotype of P. aeruginosa in some cases, such as when infection can be traced to a single serotype. However, in many other cases they are not suitable.

For example, in prophylactic treatments against latent diseases in humans, it is appropriate to administer human antibodies that are protective against multiple serotypes. Similarly, in therapeutic applications where the serotype of the infecting strain is unknown, it is preferable to administer human antibodies that are effective against most (if not all) of the clinically important P. aeruginosa serotypes. It is. A combination of human monoclonal antibodies, each specific for a single serotype of Pseudomonas aeruginosa, could theoretically be made to protect against a single serotype of P. aeruginosa. However, such a combination is
It is difficult to develop, and difficult to obtain from a manufacturing standpoint.

Therefore, there is a significant need for human P. aeruginosa monoclonal antibodies that can recognize and mount protection against multiple serotypes of P. aeruginosa. Overall, these antibodies are suitable for use as prophylactic and therapeutic treatments for Pseudomonas aeruginosa infections. The present invention fulfills these needs.

SUMMARY OF THE INVENTION Novel cell lines are provided that are capable of producing human monoclonal antibodies that can specifically react with LPS molecules of multiple (but not all) FAT & serotypes of Pseudomonas aeruginosa. . These antibodies have varying reactivity with Fisher immunotypes and bind to 0.1 or more immunotypes. Additionally, a composition comprising at least one human monoclonal antibody or binding segment thereof capable of cross-reacting with P. aeruginosa serotypes, preferably in a physiologically acceptable carrier. A method of treating a human susceptible to or already infected with Pseudomonas aeruginosa is provided. The composition may also contain any one or more of the following: reacting with other serotype or immunotyping determinants on the LPS or flagella or exotoxin A of Pseudomonas aeruginosa; additional human monoclonal antibodies that can be obtained; gamma globulins from human blood plasma; blood plasma obtained from humans that exhibit high levels of immunoglobulins that are reactive with Pseudomonas aeruginosa;
A gamma globulin fraction from human blood plasma; and one or more antimicrobial agents.

According to the present invention, there is provided a novel cell capable of producing a human monoclonal antibody and a composition comprising said antibody, said composition comprising at least a plurality of (and in some cases all) Pseudomonas aeruginosa strains. can selectively recognize P. aeruginosa, in which case individual antibodies typically
Recognizes the ATS serotype and theta, l, or multiple Fisher immunotypes. The main cells have germline DN from them.
A or precursor cells have identifiable chromosomes that have rearranged to encode antibodies (a*e-odm) that have binding sites for epitopes common among several green man serotypes. ing. These human monoclonal antibodies can be used in multi-seed formats (eg, in vivo protection), including diagnostics and therapeutics.

Typically, the cells of the invention are transformed human lymphocytes that produce protective human monoclonal antibodies against accessible LPS molecules of M. aeruginosa. By "accessible" is meant that the LP8 molecule is physiologically available in the environment used for direct interaction with the monoclonal antibody. The monoclonal antibodies thus provided are useful in the treatment or prevention of serious diseases caused by Pseudomonas aeruginosa.

LPS molecules in the outer membrane of M. aeruginosa are available for direct contact by antibody molecules, thus leading to complement-mediated lysis and (
or) facilitate phagocytosis. Furthermore, LP11 molecules that are shed from the adventitia into the surrounding environment also do not interact directly with antibody molecules and are eliminated via the reticuloendothelial system.

Preparation of monoclonal antibodies was performed using LP of various serotypes of Pseudomonas aeruginosa.
Immortal expression of nuclear sequences encoding antibodies that are specific to epitopes on the S molecule
tsm). Typically, the monoclonal antibody is produced by cell-driven Epstein-Barr virus (EBV) transformation of beta lymphocytes obtained from human donors that have been or have been exposed to the appropriate serotype of Pseudomonas aeruginosa. can get. The antibody-secreting cell line thus obtained has the characteristics of a continuously growing lymphoblastoid cell with a diploid karyotype, is positive for Nibstai/-Barr nuclear antigen, and is IgG1
secretes monoclonal antibodies of the IgG, I(IA, or IQD isotype), including various subtypes such as IQG2, IgG3, and 1gG4. This cell-driven transformation itself is described in U.S. Pat. .464.465, which is incorporated herein by reference.

This monoclonal antibody can be used directly or with Fe, Fa
b.

It can be used as a fragment (fragm-%t) such as FCab')H, but is usually used as is.

Alternatively, the antibody-producing cell line can be used in appropriate drug-marked human myeloma, murine myeloma, mouse heteromyeloma, or human) IJ
A human hybrid cell line can be obtained by cell fusion of lymphoblastoid cells with human β-lymphocytes.

The cell lines of the invention may find use other than in the direct production of human monoclonal antibodies. These cell lines are fused with other cells (such as appropriate drug-marked human myeloid deer, mouse myeloma, human-mouse heteromyeloma, or human lymphoblastoid cells) to obtain hybridomas;
Transfer of genes encoding these monoclonal antibodies can thus be provided. Alternatively,
These cell lines can be used as sources for chromosomes encoding immunoglobulins, which can be isolated and transferred into cells by techniques other than fusion. The genes encoding these monoclonal antibodies can then be isolated and used according to recombinant DNA techniques for the production of specific immunoglobulins in a variety of hosts. In particular, oD from metsenger RNA
By preparing an NA library, single-a
DNA clones can be isolated, placed into a suitable prokaryotic or eukaryotic expression vector, and then transformed into a host for final bulk production.

These lymphoblasts or hybrids can be cloned and screened according to conventional techniques.
Antibodies capable of binding to epitopes of different P. aeruginosa serotypes are detected on cell supernatants.

There are several serotyping schemes in the literature that may be used to screen cell supernatants for the presence of anti-Pseudomonas antibodies. These schemes mainly target the thermostable major antigen of this bacterium (gosα 100ca%ti
g-%) Ki Press, pp. 213-238 (1978). This multiplication of celloglue-pink schemes makes comparisons somewhat difficult in P. aeruginosa serological studies, and thus the selection of certain threads for the purpose of screening the episulcus appears to be somewhat arbitrary. . Confusion in the typing system was recently proposed by the Pseudomonadaceae subcommittee of the International Committee on Systematic Bacteriology as the backbone for future serological studies of Pseudomonas aeruginosa. This was revealed through the creation of the International Antigen Typing Scheme (IATS). This system is IATS l type, IATS
It encompasses all thermostable body antigens that have been identified in the pre-power system, which comprises 17 distinct serotypes, referred to as type Z, etc. Ryu, P.V.,,I.
*t, J, 5yat, Baatariol, 33:2
56-264 (1983), which is incorporated herein by reference.

In developing protective monoclonal antibodies that are cross-reactive between strains of P. aeruginosa, it is also advantageous to incorporate a typing scheme based on protective antigens of P. aeruginosa. The above scheme was devised with the intention of developing vaccines for clinical use and was proposed by Fisher, M.W.
et al., J.

Bactariol, 982 835-836 (196
9) is explained in detail. This system, commonly referred to as Fisher's typing thread, covers most of the known Pseudomonas aeruginosa 'I! - It is classified into seven types, called Fisher immunotype I, Fisher immunotype 2, etc. The correlation between IATS and Fisher's typing system has been determined (Liu, P.V., et al., supra) and is shown in Table 1. As noted in Table 1, each Fisher immune system corresponds to an IATS serotype, although there is no corresponding Fisher immune type for some IAT:S serotypes. I
For the ATS and Fischer typein systems, the antigenic determinants involved in the two-sided end typing scheme are thought to reside on the surface LPS molecules of P. aeruginosa (Ryu, p, v,
et al., top; Hanegene, F. et al., Natsra, 22-9
:209-210 (1979).

Table ■ Comparison and Correlation of IATS and Fischer Typing Schemes for Pseudomonas Human monoclonal antibodies are provided that are capable of specifically binding to. These determinants may be present, for example, on two or three IATS serotypes and on 0.1 or at least two Fisher immunotypes. The antibodies may be protective in vivo against some or all of the recognized serotypes and immunotypes, typically two or more serotypes.

The monoclonal antibodies of the invention may also find a wide variety of uses in vitro. For example, this monoclonal antibody can be used for typing, for the isolation of specific P. aeruginosa strains, for the selective removal of P. aeruginosa cells in heterogeneous mixtures of cells, etc. I can do it.

For diagnostic purposes, the monoclonal antibody may be labeled or unlabeled. Typically, diagnostic assays involve detection of complex formation via binding of monoclonal antibodies to LPS of P. aeruginosa.

When in unlabeled form, the antibodies find use in agglutination assays.

Additionally, unlabeled antibodies can be used in combination with other labeled antibodies (M2 antibodies) that are reactive with this monoclonal antibody, such as antibodies specific for immunoglobulins.

Alternatively, the monoclonal antibody can be directly labeled. Radionuclides, fluorophores (/J% orsacm de), enzymes, enzyme substrates, enzyme cofactors, acid accumulation inhibitors,
A wide variety of labels can be used, such as ligands (particularly haptens) and the like. Many types of immunoassays are available, and as an example, some are described in U.S. Pat.
17,827; 3,850°752; 3,90
1,654; 3,935,074; 3,984
,533;3.996,345;4,034,074;
and No. 4,098,876, all of which are incorporated herein by reference.

Typically, the monoclonal antibodies of the invention are utilized in enzyme immunoassays, where the primary antibody, or a second antibody from a different species, is conjugated to an enzyme.

When a sample containing certain serotypes of Pseudomonas aeruginosa, such as human blood or its hemolysate, is combined with a principal antibody, binding occurs between the antibody and a molecule representing the desired epitope. . The cells can then be separated from unbound reagent and a second antibody (enzyme-labeled) added. The presence of antibody-enzyme conjugate specifically bound to the cells is then determined.

Other conventional techniques known to those skilled in the art may also be utilized.

Kits can also be provided for use with the subject antibodies in detecting P. aeruginosa infection or the presence of P. aeruginosa antigens. Thus, the biological monoclonal antibody compositions of the invention can be provided alone or in combination with another antibody specific for other Durham-negative bacteria, usually in lyophilized form. These antibodies may be conjugated or unconjugated, but may be conjugated to buffers such as Tris, phosphate, carbonate, stabilizers, biocides, inert proteins, e.g. , bovine serum albumin, etc. are included in the kit. Generally, these materials are present in less than about 5% by weight, based on the amount of active antibody, and usually in a total amount of at least about 0.001 weight percent, based on the antibody concentration. It is often desirable to include inert fillers or excipients to dilute the active ingredient, in which case the excipients may be present from about 1 to 99% by weight of the total composition. If a second antibody capable of binding the monoclonal antibody is used, this will be present in the normal vial. The second antibody is typically conjugated to a label and is formulated in a manner similar to the antibody formulation described above.

The monoclonal antibodies of the invention can also be incorporated as a component of a pharmaceutical composition containing at least one therapeutic or prophylactic ii[f monoclonal antibody of the invention together with a pharmaceutically effective carrier. The pharmaceutical carrier should be any compatible, non-toxic material suitable for transporting the monoclonal antibody to the patient. Sterile water, alcohol,
Fats, waxes, as well as inert solids can be used as carriers. Pharmaceutically acceptable auxiliary agents (buffers, dispersants) can also be incorporated into the pharmaceutical compositions. The composition may contain a single monoclonal antibody so as to be specific for two or more (but not all) serotypes of P. aeruginosa. Alternatively, a pharmaceutical composition can contain two or more monoclonal antibodies to form a "cocktail." For example, a "cocktail" containing human monoclonal antibodies against a group of different Pseudomonas aeruginosa serotypes would be a general product with activity against the majority of clinical isolates of this particular bacterium. .

Of interest are at least three IATS serotypes, usually at least four, as well as more, usually at least five.
Prophylaxis and (
or) a therapeutically effective clonal antibody composition. Of particular interest are monoclonal antibody compositions that react with at least more than 7, preferably at least about 10-14, and also all 17-i (including 17) IATS serotypes. In connection with the IATS serotypes, the composition desirably comprises at least two, usually at least three, and more usually at least four, and up to and including all seven Fisher immunotyping system immunizations. Reacts with the mold.

Each of the compositions contains at least one, usually at least two
and usually at least three antibodies (in which case each antibody encompasses at least 2.3.4 or more (but not all) IATS serotypes; preferably at least two IATS serotypes and one There is at least one monoclonal antibody that binds to one or more Fischer immunotypes, preferably at least two immunotypes.

Desirably, the total number of monoclonal antibodies in the composition is at least one and the IATS with which the composition is reactive.
Less than about % of the total number of serotypes, often % of the total number.

The molar ratio of the various monoclonal antibody components is usually by a factor of 1
more than 0, more usually no difference by more than a factor of 5,
Usually there will be a molar ratio of about 1=1-2 to each of the other antibody components.

The monoclonal antibodies of the invention may be combined with other monoclonal antibody compounds or with existing immunoglobulin products such as commercially available gamma globulins and immunoglobulin products used in the prophylactic or therapeutic treatment of P. aeruginosa disease in humans. Can be used in combination with blood serum products. Preferably, blood plasma is obtained from a human donor which, in the case of immunoglobulins, exhibits high levels of immunoglobulins that are reactive with Pseudomonas aeruginosa. In general, see ``Intravenous Immunoglobulins and Combromized Hosts,'' Am, J. Mad, 76 (3).
g), March 30, 1984, pp. 1-231, which is incorporated herein by reference.

The monoclonal antibody kL of the invention can be used as a separately administered composition given in conjunction with an antibiotic or antibacterial agent. Typically, the antimicrobial agent is an aminoglycoside (e.g., gentamicin, tobramycin, etc.)
may include anti-Pseudomonas penicillins (eg, carbenicillin) in combination with P. aeruginosa, but many additional agents (eg, cephalosporins) well known to those skilled in the art may also be utilized.

The human monoclonal antibodies of the invention and pharmaceutical compositions thereof are particularly useful for oral or parenteral administration. Suitably, the pharmaceutical composition can be administered parenterally, ie subcutaneously, intramuscularly or intravenously. The invention thus provides a composition for parenteral administration consisting of a solution of a human monoclonal antibody or cocktail thereof dissolved in a usable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These compositions are
It can be sterilized by conventional, well-known sterilization techniques.

These compositions contain pH adjusting and buffering agents, toxicity modulating agents, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., which can be used as pharmaceuticals depending on physiological conditions. May contain auxiliary substances. The concentration of antibodies in these formulations is
May vary widely, i.e., from less than about 0.5%, usually 1% or at least 1%, to 15 or 20% by weight, primarily on the basis of volume reduction, particle size, etc., and preferably for the particular mode of administration selected. selected.

Thus, a typical pharmaceutical composition for intramuscular injection would include 1 m of sterile buffered water and 5 m of monoclonal antibody.
It can be configured to contain 0■. A typical composition for intravenous infusion is sterile Ringer's solution 2
50W#g, as well as the monoclonal antibody 150IR9. Actual methods of preparing parenterally administrable compositions are known or apparent to those skilled in the art and are detailed in 5 ci-%as, 15th edition, Mag Publishing Company, Easton, Pennsylvania (1980). g, which is incorporated herein by reference.

Monoclonal antibodies of the invention are lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional immunoglobulins, and known lyophilization and reconstitution techniques can be used. Freezing and reconstitution result in varying degrees of loss of antibody activity.
(e.g., in the case of commonly used immunoglobulins, IgM antibodies tend to have a greater loss of activity than IQG antibodies) and the level of use must be adjusted to compensate. It is recognized by the technical hot wire person that there is.

Compositions containing the present human monoclonal antibodies or cocktails thereof can be administered for the prophylactic and/or therapeutic treatment of Pseudomonas aeruginosa infections. In therapeutic applications, the composition is administered to a patient already infected with one or more Pseudomonas aeruginosa serotypes in an amount sufficient to cure or at least partially suppress the infection and its complications. Ru. The amount adequate to accomplish this is defined as a "therapeutically effective dose." Effective concentrations for this use depend on the severity of the infection and the general state of the patient's own immune system, but generally range from about 1 to about 200 antibodies per body weight, with 5 to 25 antibodies per body weight.
Dosages of IIg are commonly used. It must be noted that the materials of the invention are commonly used in serious disease states, ie life-threatening or potentially life-threatening situations, especially bacteremia and endotoxosis due to aerobic bacteria. In such cases, given the absence of foreign substances and the absence of "foreign bodies" achieved by the human monoclonal antibodies of the invention, it is possible to administer a substantial excess of these antibodies. , may be felt by some physicians to be desirable.

In prophylactic applications, compositions containing one or a cocktail thereof may be used to treat patients who are not already infected with Pseudomonas aeruginosa,
administered to a patient to enhance the patient's resistance to said potential infection. Such an amount is defined as a "prophylactically effective amount." In this application, the exact number of elms is generally 0.1 to 25 cm per pair, although again depending on the patient's state of health and general level of immunity.
,5 to 2.5.

Single and multiple administrations of this composition can be carried out with the dosage and type selected by the treating physician.

In all cases, the pharmaceutical formulation should provide a sufficient amount of the antibody of the invention to effectively treat the patient.

EXPERIMENTAL EXAMPLE I Example 1 demonstrates the production of human monoclonal antibodies (11M isotype) that react with IATS serotypes 2.5 and 16 and Finscher immunotypes 3 and 7.

Peripheral blood samples obtained from cystic stroma patients known to have chronic infection with Pseudomonas aeruginosa were used as a source of human B cells. Mononuclear cells were isolated from blood by standard centrifugation techniques on Ficolupac (Boiam, A.,
5 casd.

J, CJss, La&, Z*v*at, (1968) 2
1: Addendum 97.77-89), washed twice in calcium-magnesium free phosphate buffered saline (PBS).

The mononuclear cells were produced using a modified E rosette formation procedure.
- Cells removed. In summary, cells were initially incubated at 20°C at 4°C.
% lXl0' cells in PBFI containing fetal bovine serum/
- resuspended at a concentration of -. l of this suspension was then 17X
2-aminothiouronium bromide (AF) from a 10% (v/v') solution in Iscos' modified Dulbecco medium to
T) Added treated sheep red blood cells (Madsen, M. and Johnson, H.E.).

J, lmm5s, Mathoda (1979) 2
7: 61-74).

The E rosette-forming cells were removed by mixing the suspension very gently for 5-10 minutes at 4°C and then centrifuging at 2500XP for 8 minutes on a Ficolupank at 4°C. E rosette-negative peripheral blood mononuclear cells (E PBMC) forming a band at the interface
were collected, washed once with Noskov's medium, and diluted with 15% (ν/v
) FC8, L-glutamine (2 mmol/l)% penicillin (100 IU/d), streptomycin (100
μy/-), hypoxanthi:/(IXIO-'M), aminobutyry7 (4X10-7M), and thymidine (1
-6×10″sM) was pre-suspended. This medium will hereinafter be referred to as HAT medium.

Cell-driven transformation of E-PBMC was performed by co-culturing these cells with transformed cell threads.

The transformed cell threads were incubated with ethyl methanesulfonate (Eu5) of the G&15001J bacterial cell line, then 30μ
Epstadzumbar nuclear anti-a was determined by selecting in the presence of 2/- 6-thioguanine to render cells deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and thus HAT sensitive.
(A'BNA) It was a lid human lymphoblastoid cell line. This cell line is called the lA2 cell line and is
The American Type Culture Collection (A
TCC). 1A2 cells in logarithmic growth phase were suspended in mesophilic HAT medium and then combined with E PBMC at a ratio of 8 IA2 cells per E PBMC. This cell mixture was spread into eight round-bottom 96-well microtiter plates (Costar-37
99) Brate was applied to the instep, and the 7111 humid atmosphere with 6% CO2 was heated to 37°C.

By directly replacing half of the upper layer with fresh flAT medium! The food was fed on the 5th and 8th days of the plateau. Wells were observed twice a day on an inverted microscope for signs of cell proliferation. 12 days after plating, 10 of the wells
It was observed that 0% had proliferating cells in most of the wells, and the cells were dense enough for removal and testing of the cells for anti-Pseudomonas antibodies.

Enzyme linked fIBU immunosorbent assay (EL I
SA ) H technique (Engvall, AI', (1977) 5
Supernatants were screened for the presence of anti-P. aeruginosa antibodies using P. 5:193-200). The antigen plate is
Flat bottom 96-well microtiter plate (Imuroyl
Seven wells contained a variety of live cells adsorbed to the bottom of the wells. Poly-cylysin (PLL) (1μ46, flH7,2 in P'BS) was added to facilitate cell adsorption to the plastic.
) 50 μt/well was incubated at room temperature for 30 minutes.

Brush off the unadsorbed PLL, clean the plate once with PES, and add PBScP@Jyokushu suspension (0%) to each well.
7) Add 50 μt of eso-0,2) and incubate the plate for 3
It was left at 7°C for 1 hour. Unadsorbed bacteria were removed by washing the plates three times with saline-Tween (0.9% NaCl 0.05% (,/1) Tween 20). The multiple antigen plates used in this screen contained: 117 Pseudomonas aeruginosa Fischer IJ11.2 84 (ATCC
No's, 27312.27313.27315); (21 P. aeruginosa Fisher immunotypes 3.5.6 and 7 (ATCCNoa, 2'1314.273 respectively)
16.27317.27318); as well as bacteria-free microtiter.

EjLISA first blocks the plate with buffer solution [
PBSlpH7,2,5%Cw/l) skimmed am milk,
0.01% (v/w) Antiform A (Sigma), as well as 0.01% (tO/V) Thimerosal] 200
Blocked at room temperature for 60 min at μt/wael.

As a preliminary blocking step, the plate was washed three times with saline-Ly-7. Then add 0.1% Tween to all wells.
20 and 0.2% (w/,) of eagle serum albumin (B
PBS containing SA), pH 7, 2, 50 μt was added. The supernatant from the well of the J@Homare plate was replica plated into the corresponding well (50 μt/well) of the antigen plate, and the plate was incubated at room temperature for 30 minutes.

The upper groove was then removed, the wells were washed three times with saline-Tween, and the wells were treated with appropriately diluted horseradish peroxidase (ngP) conjugated anti-human rga+
Zyht (American 9 Alexis International A1114 and A1124) was added. In this example, HRP-goat anti-IQG and HRP-goat anti-IQM were combined with 0.05% Tween-20 and 0.1% E
SA? : Contains PES, pH 7, 2A, 1 each
The following concentrations were used: 1:5000 and 1:3000.

After incubation for 30 minutes at room temperature, excess t)TiFunshugate goat antibody was removed and diluted with saline-Tween for 3
The mine was cleaned. Each well was then injected with substrate 100mM citrate buffer, pH 5,000g, 8mq/me of phenylenediamine salt plus deionized H20CPo, 03%
H, O, and brating (n@equal volume mixture) 100μ
I used T as a secondary weapon. After 30 min of setup in the dark Hr, all wells had 3 N kf2S0.50μtff: Seki ≦ Katana U
The reaction was stopped by 1 yen. In the culture plate containing the antibody that reacted with the antigen of 1 note, the color development in the corresponding well was detected, and the strength of the reaction was
Quantification was determined by measuring the absorbance at 490% on a micro FILISA reader.

As a result of the 1=ilF supernatant obtained by the above method, it reacted with Fisher immunotype 3.5.6 and 7 plates, but it reacted with Fisher immunotype 1.2, as well as 4 plates or germ-free plates. Two wells were identified that contained no anti-P. aeruginosa antibodies. To identify specific Fischer immunotypes, antigen plates bearing only one Fischer immunotype PLL-fixed bacterium were prepared as described above for each immunotype. Performance of the ELISA assay as described above using culture slides from wells IC'l and 2H12 on individual immunotype plates.
s, showed that these two wells harbored antibodies specific for both Fisher immunotypes 3 and 7. Pseudomonas aeruginosa typing strain (obtained from ATCC, Net, 3
I A 1'S of 3348-33364Y'ffmu)
A similar ELISA performed on panel well lC1
and 2H12rP antibody has IATS 2.5 and 16
It was clearly shown that there is a specific reaction with

Wells IC1 and 2H12 A-reactive antibody inotypes were HRP-goat anti-human IQG and HRP-goat anti-human I
QM can be used independently as a second step reagent rather than in combination.
The specificity test was determined by an ELISA assay similar to the specificity test described above, except that it was used as a ELISA assay. Positive reactions of well IC1 and 2H12 A antibodies by Fisher immunotypes 3 and 7 were detected only in the case of anti-1gM reagent,
The 19M intib was specified for the antibody produced by Tamotsu Yasuyoshi. The anti-Fisher immunotype 3/i and 7 reaction pattern is well-defined with one or more antibodies that are reactive with IGI and 2H12 (i.e., one or two antibodies that are reactive with both Fischer immunotype 3 and 7, Fisher immunotype 3). cells from both wells were subcultured at a lower density to determine whether the cells from both wells were subcultured at a lower density and the wells containing proliferating cells. were assayed for antibody activity on separate Fisher immunotype 3 and Fisher immunotype 7 bacterial antigen plates. Subcultures of H lacking the aminopterin component
A total volume of 100μ of AT medium (HT medium) was plated in a 96-well round bottom plate at a density of 5 cells/well. Non-transformed, HAT-sensitive lymphoblastoid cells were included as feeder cells in all wells at a density of 500 cells/well. Four days after blating, 100 μt of HAT medium was added to all wells to selectively kill feeder cells. Half molecule HA in supernatant 9 days after blating
Wells were re-fed by replacing with T medium. Thereafter, the wells were similarly fed with HT medium every 4-5 days until the wells had sufficient lymphoblastoid cells for supernatant analysis by ELISA. Individual Fischer immunotype 3 and Fischer immunotype. When assayed on 7 antigen plates, all supernatants that reacted with Fisher immunotype 3 also reacted with Fisher immunotype 7, and 1
One antibody was shown to be responsible for activity against both Fisher immunotypes. Randomly selected supernatants were IAT
When assayed on S strains 2.5, and 16, one antibody was found to react with all three strains but not with individual strains, and one antibody was found to react with Fisher immunotypes 3 and 7 and I
This further supported the conclusion that it was cross-reactive with ATS strains 2.5, as well as 16.

Cloning of specific antibody-producing cells from wells 1C1 and 2H12 showed that all clonal supernatants assayed by the ELIEIA protocol above were associated with Fisher immunotypes 3 and 7 and IATS bacteria 2.5, and 16.
This was done by subjecting cells from each well independently to several rounds of limiting dilution cloning until a positive reaction occurred. Cloning used feeder cells as described above for subcultures. By these means, serial (mortal) and Fisher immunotypes 3 and 7 and IATS strains 2.5, and 1
Two clonally transformed human cell lines secreting human monoclonal antibodies reactive with 6 were achieved. In this example, the cell line and the antibody it produces have the same name (dasig%atio%).

Example 1 Example 1 shows a human monoclonal antibody (I
The method for producing IG isotype) is clearly demonstrated. The protocol for production is essentially the same as in Example 1. In summary, a tumor was obtained from a patient with fibrosis known to have chronic infection with Pseudomonas aeruginosa. peripheral blood sample)
[Used as a raw material for ll1ffa. PBS suspension 1.
6- was used with a 1.6×10 AET-treated sheep red blood cell suspension.

Monoclonal cells were isolated as described in Example 1.

Cell drive transformation was the same except for the following fish: the ratio of IA2 cells per E-PBMC was 7.5; Cultures were fed on days 6 and 10 after plating; and on day 16 after plating, almost all of the wells contained growth channels.

Screening of culture supernatants for specific antibodies was performed as described above and resulted in Fisher immunotype 3.
One well (9D1) containing antibodies that are reactive with 5.6, and 7 plates, but not with Fisher immunotype 1.2 and 4 plates or asterile plates.
decided on the location. The ELISA assay described above on individual immunotype or channel-type bacterial antibody plates using 9Dl culture supernatants was performed on both Fisher immunotypes 3 and 7, as well as on IAT.
Antibodies specific to S strains 2.5 and 16 were shown. Subsequent studies performed according to Example I demonstrated that the percent antibody isomerism in well 9D1 was attributable to a single clonally secreted IgG isotype antibody.

EXAMPLE ■ Example ■ demonstrates the antigen specificity of some of the antibodies of the invention. Monoclonal antibodies IC'l, 2H12 and 9
Additional assays were performed to determine whether Dl reacted with the same antibody label and thus the specificity was the same. First, the antibodies were compared in an ELISA on a panel of control and clinical isolates of P. aeruginosa. The ELISA protocol was as described above with the following modifications: 1) Instead of bacteria adsorbed to PLL-coated plates, the wells of the plate contained whole bacteria fixed in ethanol at the bottom of the wells. . The plate contains PB into the wells.
Addition of 50μ of washed bacterial suspension (OD, 6° = 0.2) in S, centrifugation of the plate for 20 min at 500XP,
Prepared by aspiration of PBS, addition of 75 μt ethanol for 10 min, removal of ethanol, then air drying. IATS strains 2.5.11 and 16 (ATCC No. 5. 33349.33352, respectively) on antigen plates.
33363) and Difco according to the manufacturer's instructions.
Detroit, Michigan] 16 clinical cases previously typed with serotype 2.5.16, as well as combinations of said serotypes, by agglutination and ELISA (as specified) using Bacto P. aeruginosa antiserum. isolates were included; 2) rabbit typing antisera were used in PB except for anti-IATS-16, which was used at a dilution of 1:250;
It was used at a dilution of 1:500 in S. lC1,2H
Culture channels containing 12 and 9D1 antibodies were used neat; 3) Biotinylated protein A (B-2001, Vector Laboratories, Inc., Birmingham, CA) diluted 1:500 as second step reagent; and 1:500 diluted biotinylated goat anti-human IyC4703 (Tavo, Inc., Burgingham, Calif.) were used for detection of rabbit and human antibodies, respectively. Add 50 reagents to appropriate wells.
μt was added and after a 30 minute setup at room temperature, unbound reagent was removed and the wells were washed three times. Next, preformed adenine:biotinylated horseradish peroxidase complex (Pectastein AB
C kit, Vector Laboratories, Inc., Birmingham, CA). After 30 minutes of apparatus at room temperature, excess vector stein A
The EC solution was removed and the wells were washed three times before addition of substrate.

As shown in Table H, the results of this assay showed that antibody I
We showed that C1 and 9Dl reacted with any clinical isolates typed as IATS 2.5 or 16 serotypes, whereas antibody 2H12 did not react with the three said isolates.

This indicates that antibody 2H12 recognized a different epitope than that recognized by IC1 or 9D1, and that these two epitopes were associated with IATS serotype 2.5,
Alternatively, this suggests that most (but not all) of the clinical isolates corresponding to 16 are expressed in a synchronized manner.

This data further shows that the molecular targets recognized by antibodies IC1 and 9D1 are IATS serotypes 2.5 or 16.
Although it appears to be present on all clinical isolates of P. aeruginosa that could be typed as belonging to P. aeruginosa, it was suggested that the target recognized by antibody 2H12 is expressed on a subgroup of said isolates.

Were the IC1, 2H12, and 9D1 antibodies reacted with different antibodies or, alternatively, the same epitope bearing the epitope was expressed in most (but not all) of the IATS 2.5 and 16 serotypes? An immunoplot assay was performed to determine the IATS2
．． The divided antigenicity between 5, as well as 16 appears to be for thermostable antigens (Liu, P. V., et al., supra), and thermostability was previously noted. It is said to be a property of lipopolysaccharide! $ According to the truth, IA
LPS preparations from 'lS strains 2.5.16° and 11 were selected as antigen preparations for analysis. The crude LPS is
prepared by extraction in saline at 60°C from the type strain (Orskot, F. La., 79:142-15
2). LP810 as determined by 2-keto-3-deoxyoctonate (KDO) content from each of the serotypes.
μ2 was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10-20% gradient gel CEDE-PAG.
E> (hankosuku, R, E, W, and curry, A, &,
, J. Bact*rjoj, (1977) 140:90
1 to 910). Separated molecular species were determined as described elsewhere (Tobin, Ho et al., Proc., Na.
tl, Acad.

Sci, USAC1979) 76:4350-4354
) The gel was transferred to a nitrocellulose membrane (NCM) and N
CA (7' loft fPBs - blocked for 1 hour in tween (Batiger, B, et al., J, lmm5cno
1. Math, (1982) 55:297-307).

Plots were then incubated for 1 hour at room temperature in 20 g of spent culture supernatant from IC1, 2H12, or 9D1 cell lines. PBS-Tween 95 minute wash L NCId
Each of the plots was analyzed using a 1:1000 or 1:1500 dilution (FEE
- in Tween) for 1 hour at 25°C.

The plots were then washed with PBS-Tween 9515 for 15 min and nitroblue tetrazolium 15-promo 4-chloro-3-indolylphosphate (NE) as described by (1983) 80:4045-4059.
Antigen-antibody interactions were visualized by incubating the T-BCIF) substrate 30-medium plot at 25° C. for 15-20 minutes. Color development was stopped by washing the plots several times in deionized water.

The profiles of the plots obtained with the three antibodies were significantly different. Antibody 2H12 was serotype 2.5, as well as regularly spaced in 16 antigen preparations (i.e.
ladder-like) preferentially recognized short sequences of low molecular weight molecules. A few regularly spaced crystalline door molecules were also slightly recognized. No reaction on the LPS preparation from serotype 11 was observed. Repeated immunoplot analyzes using LPS preparations previously treated with brotainase at 60° C. to destroy protein antigens did not change the profile.

The low molecular weight bands indicate that the antigen was not transferred to NCH and was instead transferred to LPS, except that the lowest band (representing lipid A of the core region plus LPA) was not recognized on the silver-stained gel.
stained specifically for the presence of (Tsuai, C,M,
and Flash, C.E., Asat, Biochgr.
(1982) 119:115-119) in a similarly obtained 5DS-PAGE gel, closely matching the smaller form of 528 molecules. Antibody I
C1 recognized the same series of regularly spaced low molecular weight molecules between serotype 2.5, as well as 16 (but not 11) antigen preparations, but the strength of the reaction was lower than that of 2H12.
In this case, it was not strong-rooted. However, in addition, this antibody also preferentially recognizes serotypes 2.5, as well as a series of relatively-molecular weight molecules regularly spaced over 16, which indicates that the relatively low molecular weight molecules recognized are Combined with the Molecular Bon Band,
Different total ladder strain profiles resulted. These profiles were not altered by pretreatment of the antigen preparations with Brotainase K. Again, these profiles corresponded to the ladder-like banding patterns observed in LPS-specific stained gels, except that no band representing core plus lipid A was recognized (i.e., they ). Antibody 9D1 has an IATS of 2.5 and 16 (but not 11)
It had the same reaction profile as the anti-strain antibody IC1.

Again, the lowest band of the ladder of silver-stained gels of the different LPS preparations was not recognized and the overall profile was unchanged by pretreatment of the LPS preparations with proteinase K.

Taken together, these observations support serotypes 2.5, as well as 1
LPS of 6 was the molecular target recognized by 2H12, IC'l, and 9D1 antibodies.

Example ■ Example ■ demonstrates the protective activity of one of the antibodies of the invention.

In order to evaluate the in vivo protective ability of antibody IC1, animal defense studies were conducted in mice. IC1 antibody was first concentrated from spent culture supernatants by precipitation with saturated ammonium sulfate (50% final 4°C) (Gud, A, H,
et al., 5 selected Methods in Ca
lha clar imlmm5nolo.

Mitschel, B.B., and Shiigi, S.M., eds., W.J.
, Freeman & Company, San Francisco, California, 279-286 (1980)). the precipitated material.

Reconstituted in a minimal volume of sterile water, extensively dialyzed against PBS, and sterile filtered. As a negative control, IATS strain 1
Another transformed human cell line (6F11-ATCC) that produces human monoclonal antibodies specific for LPS
Spent culture supernatant from ACRL8562) was treated similarly.

Female EALB/c mice weighing between 20 and 229 were divided into two groups of 13 mice each. All mice in each group were individually injected with concentrated 1C1 or 6F11 antibody 0.5 - i.p.
p) Inoculated by route. After 6 hours, each of the two groups'i
Divided into 3 groups of 10 mice, members of each group of 10 mice were individually treated with IATS strains 2, 5,
Or 11 5LD. Nakan@turbidity containing 0.3 d
I did an IP challenge.

Bacterial suspensions were prepared from pre-logarithmic Pros cultures, from which the bacteria were centrifuged, washed twice in PBS, and resuspended to approximately the same density in PBS. Control groups of 4 or 5 mice each were injected intraperitoneally with PBSo, 5m1f, and 6 hours later challenged intraperitoneally with the same serotype (5LDw). Following bacterial challenge, animals were observed for 5 days.

As shown in Table IIIK, antibody ICI
and IATS5, but not IATS11 serotype, produced specific and significant protection against lethal challenge.

The typical course following bacterial challenge in all unprotected animals was endotoxic shock of variable duration, usually leading to death. Control animals given only PES underwent acute internal shock and all rapidly died.

Negative control animals given the non-homologous antibody (6F11) had a relatively long period of shock characterized by the symptoms listed in footnote @a''' of Table m. Some of these animals , possibly due to concentrated non-antibody components in the antibody preparation, eventually recovered. However, animals given protective homolobed antibodies displayed only slight symptoms of endocytosis. Symptoms disappeared within 24 hours of inoculation, at which time the animals appeared healthy for the remainder of the 5-day observation period.

Table ■ In vivo demonstration of the protective effect of human monoclonal antibody IC1 against IATS serotypes 2 and 5 IcI 8/1
0 10/10 3/10"6J'll
O/10 1/10' 10/10PBS
O/4 0/4 0/4a, if non-specific protection is observed, recovery from infection is due to the interaction between homopg antibodies and the infecting strain, i.e. IC11 in case of IATS 2 or 5 or IATS 11 Case 6F11 was significantly different from that seen. In the latter case, recovery was essentially complete 24 hours after bacterial challenge and the animals appeared normal. However, in non-specific cases, mouse strains exhibit signs of acute infection (
(diarrhea, eye crusting, piloerection, "hunt-type" profile, and slow behavior). The non-specific protection is likely due to non-antibody components that are co-concentrated and thus injected into the animal. This is because all animals given PEI alone died.

A second experiment was conducted using the same protocol in which groups of mice were challenged with Fisher strains 2.3 or 7 and animals were observed for 5 days. As shown in Fig. 1v, antibody 1C1 again produced specific and significant protection against lethal infection of Fisher immunotypes 3 and 7, but was ineffective against Fisher immunotype 2.

Table ■ In vivo evidence of the protective effect of human monoclonal antibodies against Fisher immunotypes 3 and 7 (:'1 10/
10 10/10 1/1066F11 0
/10 7/10 10/10PBS
O15015015 survival is likely due to non-specific protection as explained in the footnote to Table ■.

EXAMPLE V Example V&C demonstrates the preparation of human monoclonal antibodies that react with IATS serotypes 4 and 11 and Fischer immunotype 2 of Pseudomonas aeruginosa. The antibody described in this example was used in a single cell,
The methods described in Examples 1 to 2 were repeated, except for the fish that needed to be subjected to several (15) decorations to characterize and assay. Results obtained for monoclonal antibodies.

The raw material for human B cells is a low molecular weight polysaccharide preparation isolated from Fischer immunotype 3 (Beer, G, B, et al., Infme, Imtycsn, C1984) 45:
309-313, herein incorporated by reference)).

Cell-driven transformation of EPBMC was achieved by co-culturing these cells with transformed cell lineage IA2. HAT 1,42 cells in logarithmic growth phase
Suspend in medium, then EPBMC2b or 15 IA
Combined with E PBMC at a ratio of 2 cells. This cell mixture was placed in 14 microtiter plates at 62,0
200μ per well at a concentration of 00 cells/well
Brating was carried out at a capacity of t. Cultures were fed on days 7 and 11 after blating, and by day 15 100% of the wells were observed to contain proliferating cells.

To screen for the presence of anti-Pseudomonas aeruginosa antibodies, two antigen plates were used consisting of = (
1) Pseudomonas aeruginosa Fisher immunotypes 1 to 7 (ATC
CNon27312.27313.27314.273
15.27316.27317 and 27318); and [(2) PLL-treated microtiter without bacteria.

Analysis of culture supernatants according to the above example method resulted in approximately 100 wells containing anti- P. aeruginosa antibodies that were reactive with Fisher immunotypes 1-7 plates but not with PLL-treated plates lacking bacteria. was identified. To identify the specific Fisher immunotypes recognized, antigen plates were constructed as above, with each row of the plate containing only one Fisher immunotype PLL-identified bacteria. Culture supernatants from each of the anti-P. aeruginosa positive wells placed in a cylindrical array on a fresh antigen plate were used to perform ELIS as described above.
As a result of performing A, a number of wells containing Fisher immunotype 2-specific antibodies were identified. To further analyze the serological specificity of anti-Fischer immunotype 2 antibodies,
A similar ELI on antigen plates configured to contain each of the 17 IATS serotypes of P. aeruginosa in separate wells.
Supernatants were tested in SA.

The results of this assay show that the majority of the supernatants are specific for IATS serotypes, but one supernatant is specific for IATS serotypes 4.11.13, as well as 14 in well 6D6. It was shown that the sexual pattern is clear.

Addition of supernatant from well 6D6 to this anti-serotype IATS 4.11.13, as well as 14 to determine whether the reaction pattern is due to one antibody or to multiple antibodies in well 6D6. Samples were tested separately at IATS4.
7 (negative control), 11.13, and 14,
The adsorbed supernatant was then subjected to five IAT! assays according to the ELISA assay outlined above. ; each of the serotypes was tested on PLL-fixed bacteria.

Adsorption was performed by resuspending 4 sets of packed microbial cells with an equal volume of supernatant on ice for 0.5 h and then separating the supernatant from the bacteria by centrifugation. The results of this assay are shown in the IATS dish ff! The ability of 4 and 11 to adsorb antibody activity to each other i, IATI dish clear type 7.13,
Alternatively, it was shown that 14 was not adsorbed. This demonstrated the presence of at least two different anti- P. aeruginosa antibodies, at least one of which cross-reacted with IATS dish types 4 and 11, in well 6D6.

Isolation and cloning of excess antibody-producing cells from well 6D6 was performed in three steps. The first step involves low-density subcultures of cells in 3 at 20 cells/well;
20 cells/r:) 1 of 9 low-density sub-cultures (5 cells/well) of cells obtained from anti-IATS serotype 4+11-positive wells generated during culture.
This was followed by nine seconds. Each 9-nd sub-culture contains a total of 10
Tests were carried out in 96-well round bottom plates at the stated density in HAT medium (HT medium) lacking 0 μt of the aminopterin component. Non-transformed HAT-sensitive Sumpablastic axons were included as supporting cells at all densities of 500 cells/Wael. Four days after blating, HAT medium was added to all wells to selectively kill the supporting cells. Wells were re-fed 9 days after blating by replacing half of the supernatant with HAT medium. Thereafter, 9 wells were similarly fed every 4-5 days with HAT medium until the wells had sufficient lymphoblastoid cell density for supernatant analysis by ELISA as described above.

In each assay, supernatants that were reactive with IATS serotype 4 were also reactive with IAT; serotype 11 and Fischer immunotype 2. Besides, antibody activity against IATS dishes m types 13 and 14 was lost, confirming the previous specificity assignment.

This formal cloning of specific antibody-producing cells involves l&?
This was done by plating the /J cells at a very low density (1/well) in a volume of 10 pt/well into 72-well Terasaki plates (8%% 61-36538). Plates were placed in an incubator for 3 hours to allow the cells to settle to the bottom of the plate, then wells containing single cells were scored microscopically for two different individuals.

Each of these cells was then separated into 9 cells along with rfa cysts.
The cells were cultured in 617-well round bottom plates as outlined above for low density subcultures. When assayed by the ELISA protocol above, supernatants from all represented clones showed anti-IAT! One dish was positive for f#m4 and 11.

By these means, the cloned transformed human #J secretes continuous Km5 [highly immortal] and human monoclonal antibodies reactive with Fisher immune u2 and cross-reactive with FAT8 serum ti4 and 11. The cell filaments were collected in succession. In this example 3, the ld cell line and the antibody it produces have the same designation (ie, 6D6).

Well 6D6 antibody inotypes were HRP-goat anti-human) 1gG and HRP-goat anti-human) IgM, which were used separately as second step reagents rather than in combination. It was determined in an ELISA assay similar to 8. Fischer immunotype 2 and IA
A positive reaction of the antibody in well 6D6A with TS dish types 4 and 11 was observed only with anti-IQM reagent, demonstrating the 19M isotype for this antibody.

The biochemical properties of the molecular species recognized by the 6D6 antibody are Fisher immunotype 2 and IATS dish types 4 and 11.
An LPS preparation from 2000 was selected as the antigen preparation for analysis, which was performed by immunoplot analysis as described in Example 3 above. LP from Fisher immunotype 1
S preparation was included as a negative control.

Prior to analysis, each of the crude LPS preparations was diluted with dissociation buffer C0,
125M Tris, 4% (w/w) sodium dodecyl sulfate (SDS), 20% (w/w) glycerol, 1
0% (,/Kan) β-mercaftethanol, 0.4% (
w/υ) Fromphenol blue, pH 6, s] Ko1=
1 dilution and bath sonicated for 6 minutes. To degrade proteins in X, proteinase KCH2c) LIPl m
y/mz) was added to each of the samples at a 40% (w/w) ratio of the enzyme mixture to r, ps and incubated at 60°C for 2 hours.
After 1 hour, bath sonication was performed for 5 minutes. Next, the sample was heated to 100°C.
The mixture was heated for 5 minutes and centrifuged in a microfuge for 2 minutes.

Clear samples representing 10 μy of LPS from each of the bacterial strains were subjected to 2S SDS polysaccharide gel electrophoresis (sns-pAax) as described above. The separated molecular species are N
Transferred to CM, NCM was incubated for 2 hours at room temperature for 3 hours on a fine culture of 6D6#I cell system. The remaining operations were as described above.

Positive milled rice ↑ζ fat 7-year non-throw type 2. It was observed only in tracks A containing IATS dish type 4 or IATS dish ff dish type ft type 11S. In tracks with Fisher immunotype 2 and IATS serotype 11,
Antibody 6D6 was a short series of regularly spaced (
That is, the lowest band on the silver-stained gel (representing the core region of LPS plus libido A) was not recognized outside the point plate.
A similarly preformed 5DS-
A relatively small form of LPS made available during PAGE
It matched the molecule precisely. In contrast, in the lane containing IATS serotype ALPS, antibody 6D6
observed a whole series of regularly spaced bands extending almost the entire length of the gel, with the strongest reaction occurring among the relatively polymeric bands. Again, this profile corresponded to the ladder-like banding pattern observed in LPS-specific staining gels, except that no band representing core plus libido A was recognized (i.e., they were paired with bands). It appeared that the band would respond).

These data clearly demonstrated that the O-side chain of LPS is the molecular target recognized by antibody 6D6 on Fisher immunotype 2 and IATS serotypes 4 and 11.

To evaluate the in vivo protective potential of antibody 6D6, animal protection studies were performed in mice. 6D6 antibody was first concentrated from spent culture supernatants by precipitation with saturated ammonium sulfate (50% final concentration). The precipitated material was reconstituted in a minimal volume of sterile water, thoroughly dialyzed against PBJ,
Sterile filtered. As a negative control, Fisher immunotype 1
Another transformed human cell line (C5B7-ATCC/
Spent culture supernatant from 16CRL8753) was treated similarly. Similarly, as a positive control, Fisher immunotype 2
and a transformed human cell line 6J' producing human monoclonal antibodies specific for LPS of IATS serotype 11.
Spent culture supernatant from l 1 (ATCC rot CRL 8562) was reduced to zero.

Female Swiss-Webster mice weighing 20-2290 were divided into 3 groups of 20 mice each. Concentrated 6D6, C3B7, or 6F11 was administered to all mice in each group individually.
Antibody 0.5- was inoculated by intraperitoneal (ip) route.

After 6 hours, the three groups were subdivided into 4 groups of t-5 mice each, and members of each group of 5 mice were treated with Fisher immunotype 1% Fisher immunotype 2, IATS serotype 4, respectively.
Or 8LD of IATS serotype 11! ip challenge with a live bacterial suspension containing 0.3-. Bacterial suspensions were prepared as described above in Example II. After the bacterial challenge,
Animals were observed for t-5 days. The results were as follows: Antibody 6D6 showed specific and significant activity against lethal challenge of Fischer immune Rm2, IAT:S fR type 4, as well as IATS serotype 11. Protection was obtained, but not with Fisher-immunized Wl.

Example ■ Example ■ is IAT for Pseudomonas aeruginosa! A method for producing human monoclonal antibodies that react with serotypes 6 and 13 and Fischer immunotype 1 is demonstrated. In order to isolate, characterize and assay the antibodies described in this example, the methods described in Examples 1-V were repeated except for fish, which required some modifications. Below are the results that can be observed using the engineering modifications and monoclonal antibodies described herein.

The raw material for human B cells was a high molecular weight polysaccharide preparation isolated from Fischer immunotype 2 (Beer et al., hge-C
1Immms, 34:46,1 (1981)). The two mentioned above, E”-
After collecting PBMC, 10% (−
/f) Cells were frozen in FC8 containing DMSO.

After these cells were harvested, they were quickly thawed at 37° C., washed once in Iskot's medium, and suspended in HAT medium at room temperature.

Cell-driven transformation requires 15 cells per E″″PEMC.
A LA2-to-cell ratio of 2 was performed.

This cell mixture was plated into 20 microtiter plates at a concentration of 78,500 cells/well. Cultures were fed every 3-5 days after blating, and on day 11, 100% of the fers were observed to contain proliferating cells.

To screen for the presence of anti-Pseudomonas aeruginosa antibodies using ELISA technology, antigen plates were prepared with Pseudomonas aeruginosa Fisher immunotypes 1-7 [respectively clinical isolate PSA1277].
(Genetik Systems Corporation Organism Bank (G5C0E''), ATCC 273
13, PSA09B (GSCOB), ATCC273
15, PEAF625CGF; C0B), ATCC27
3L7 and ATCC27318). Bacteria-free PLL-treated microtiter plates were also used in this screen.

Analysis of the culture supernatant using the above method revealed that it was reactive with Fisher immunotype 1-7 plates but lacked bacteria.
Approximately 200 wells containing anti-Pseudomonas aeruginosa antibodies that were not reactive with the L-treated plates were identified. 2 or more IA
In order to identify 9ELs containing antibodies reactive with TI hematopoiesis, antigen plates containing PLL immobilized cells of the FAT8 serotype were created, with only one column of the plate as described above. Ta. ELISA was performed as described above using supernatants from trough expansion cultures of anti- P. aeruginosa positive wells. Transfer the supernatant to a new antigen plate in one row.
As a result, a number of wells containing antibodies reactive with many IATS serotypes were identified. One well, designated 8H7, contained antibodies specific for IATS dish types 6 and 13.

When the supernatant from this well was tested by 7 Fisher immunotype ELISA as in Example V,
The 8H7 antibody was demonstrated to be specific for Fisher immunotype 1.

To determine whether the anti-IA'1SffiL serum types 6 and 13 reaction pattern is due to polyphasic antibodies in well 8H7A, an additional aliquot of the supernatant was separately IATS blood 1''
Type #6.13, as well as 17 (negative control) were used for adsorption, and the adsorbed supernatant was then subjected to EL as outlined above.
PLL sessile bacteria of each of the three FAT & serotypes were tested according to the ISA assay. The result is IATSI
The O1 clear type showed that antibody activity was adsorbed to the calf. IA
TSMri serum type is anti-r AT S dish TWm6 or 1
No antibody activity was adsorbed to any of 3. This data demonstrated that the reaction pattern in anti-IATS dishes 6 and 13 was due to a single antibody from well 8H7.

Isolation and cloning of antibody head somatic cells from well 8ff7 was performed essentially as described in Example V in three steps. By these means, cloned and transformed cell lines that are continuously viable (i.e., immortal), reactive with Fisher immunotype 1, and cross-reactive with IATEI serotypes 6 and 13 are achieved. It was done. In this example, the cell thread and the antibodies it produces have the same designation, 8H7. Using a procedure similar to that described in Section A, the isotype of the pile body in well 8H7 is
It was determined that gM.

Biochemical characterization of the molecular species recognized by 8H7
LPs from Fisher immunotype 1 and IATEi serotypes
The S preparation was chosen as the antigen preparation for the analysis, which was performed by eight iminoplots as described in Examples Ⅰ and ⅲ above. LPS preparation from IATS dish ffI type 10 was included as a negative control.

The fraction of the obtained iminoplot was determined by Fischer immunotype 1% IATS dish type 6 or IATS dish type 13 L.
Positive results were shown only in NCM tracks containing PS.

In the Fisher immunotype 1 and IATS dish m6 tracks, antibody 8H7 recognized a series of regularly spaced (ie, ladder-like) bands that spanned nearly the entire length of the gel. These bands (engineering) were also performed on a 5ns-PAGE gel in which the antigen was
The polymolecular form of the LPS molecule corresponded to the 4-fold duct, as was visualized in the H. but not specifically stained for the presence of LPS). IATSmff
In the type 4 13LPS 3L rain, antibody 8H
More shortened regularly spaced bands were recognized in the medium to upper molecular weight range of the 7Gζ glue. Again, the highest and lowest molecular weight forms of the stained gel did not appear to be well recognized during Western plots outside the fish flesh.
The next band recognized corresponded in position to that observed in the LPS-specific staining gel. These data are
It was clearly shown that LPS is the molecular target recognized by antibody 8H71/C on Fisher immunotype 1 and IATS serotypes 6 and 13.

In order to evaluate the in vivo protective ability of antibody 8H7, the above example ■
Animal protection studies were performed in mice as described in V. and V. As a negative control, one other transformed human cell line (6JF'1l--ATCCNo, CRL
8652) from spent culture supernatant (Fisher immunotype 2
(which produced human monoclonal antibodies specific for LPSIIC) were treated similarly. As a positive control, the transformed human cell line C3B7 (ATcc locus CRL
Spent culture supernatants from 8753 (which produces human monoclonal antibodies specific for Fisher immunotype 4 and IATS serotype 6) were used.

Female Swiss-Webster mice weighing 20-27 J were divided into 3 groups of 30 mice each. All mice in each group received either concentrated 8H7, 6F11, or C2H4 antibody 0.0.
5rnl was inoculated intraluminally (1p). After 4 hours, each of the three groups was subdivided into three groups of 10 macus, and members of each group of 10 macus were given IATEi6 serum! 9.4LD of a clinical isolate (A522) representing (Fisher immunotype 1 equivalent). , IATS13 reference blood type 5
L.D. , or IATSll (Fisher immunotype 2
equivalent) 10LD of the reflex 7 Lance serotype. A live bacterial suspension containing o, aILt was challenged ip.

The IfllVi suspension was prepared as described above in Example II. Following bacterial challenge, animals were observed for 5 days.

The results were as follows: Table ■ In vivo demonstration of the protective effect of human monoclonal antibody 8H7 against IATEI serotypes 6 and 13 8H79/10 6/10 1/10 C5E 7 9/10 0/10 0/ 106F11
0/10 2/10 10/10 As shown in Table ■, antibody 8H7 provided specific and significant protection against the IATS Serum Castle 6 lethal challenge, but IATEI
blood? # It was not obtained with type 11. IATS dish ffm1
LD for this isolate when compared to the dish #Iu6 isolate, although to a lesser extent protection against 3. This may be explained by the relatively large number of bacteria required to achieve this (&3 x 10' colony forming units and 2.6 x 10' colony forming units respectively).

In another experiment: In one experiment, antibody 8H7 was
3.5LD of isolates. 5 out of 5 mice challenged with IATS6 isolate and 5 out of 5 trout challenged with IATS6 isolate were protected.

From the foregoing, it can be seen that the cell lines of the present invention are derived from various Pseudomonas aeruginosa I
AT:! It has been observed that cross-reactive human monoclonal antibodies and fragments thereof are produced. This allows relatively easy development of prophylactic and curative compositions that are likely to be effective against most (if not all) strains of Pseudomonas aeruginosa. Additionally, these cell lines produce antibodies that have utility in immunoassays and other well-known procedures.

Although the invention has been described in some detail by way of illustration and for clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the claims.

Patent applicant Genetic Systems Corp. V-
John

Claims (1)

Claims: 1. A composition comprising human monoclonal antibodies or binding fractions thereof capable of specifically binding to several, but not all, IATS serotypes of Pseudomonas aeruginosa. 2. The composition of claim 1, wherein said antibody is in vivo protective against at least two IATS serotypes. 3. The composition of claim 1, wherein said antibody is in vivo protective against three IATS serotypes. 4. The composition of claim 1, wherein said antibody is capable of binding to two to three IATS serotypes. 5. The composition of claim 4, wherein said antibody is in vivo protective against at least two IATS serotypes. 6. The composition according to claim 1, wherein the antibody is non-reactive with any of the Fisher immunotypes of Pseudomonas aeruginosa. 7. The composition of claim 1, wherein the antibody reacts with one Fischer immunotype of Pseudomonas aeruginosa. 8. The composition of claim 1, wherein said antibody is capable of reacting with two or more Fischer immunotypes of Pseudomonas aeruginosa. 9. The composition of claim 8, wherein said antibody is capable of reacting with three IATS serotypes. 10, the antibody has at least two IATS serotypes and 1
8. The composition of claim 7, which is protective in vivo against two Fischer immunotypes. 11. The composition of claim 8, wherein said antibody is in vivo protective against at least two IATS serotypes and at least two Fisher immunotypes. 12, comprising at least two human monoclonal antibodies, at least one of which has an accessible lipopolysaccharide determinant present on less than all, but at least two, IATS serotypes of Pseudomonas aeruginosa; A composition capable of specifically reacting. 13. At least one of the antibodies has two or more IA
13. The composition of claim 12, which is in vivo protective against TS serotype. 14. The composition of claim 12, wherein said antibody is in vivo protective against three IATS serotypes. 15. The composition of claim 12, wherein said antibody is capable of binding to three IATS serotypes. 16. The composition of claim 12, wherein the antibody is non-reactive with any Fischer immunotype. 17. The composition of claim 12, wherein said antibody is capable of reacting with one Fischer immunotype of Pseudomonas aeruginosa. 18. The composition of claim 12, wherein said antibody is capable of reacting with two or more Fischer immunotypes. 19. Claim 16, 1, wherein the antibody is in vivo protective against IATS serotypes and Fisher immunotypes.
7. The composition according to item 18. 20. An antibody or binding segment thereof, wherein the antibody or binding segment thereof is capable of specifically reacting with multiple but not all IATS serotypes and less than or more than one Fischer immunotype of Pseudomonas aeruginosa. A composition consisting of: 21. The composition of claim 20, wherein said antibody or segment thereof is capable of reacting with at least three IATS serotypes of Pseudomonas aeruginosa. 22. Claim 20, wherein said antibody or segment thereof is capable of reacting with two Fischer immunotypes of Pseudomonas aeruginosa
Compositions as described in Section. 23. The composition of claim 20, wherein the antibody is in vivo protective against at least two IATS serotypes and at least two Fisher immunotypes. 24. The composition of claim 20, wherein the antibody reacts with IATS serotypes 2, 5, and 16, and Fisher immunotypes 3 and 7. 25. A composition comprising an antibody or segment thereof, wherein the antibody or segment thereof is capable of specifically binding to multiple, but not all, IATS serotypes and one Fischer immunotype of Pseudomonas aeruginosa. 26. The composition of claim 25, wherein said antibody or segment thereof is capable of binding to two IATS serotypes. 27, the antibody has at least two IATS serotypes and 1
26. The composition according to claim 25, which provides in vivo protection against two Fischer immunotypes. 28. The composition of claim 25, wherein said antibody reacts with IATS serotypes 4 and 11 and Fisher immunotype 2. 29. The composition of claim 25, wherein said antibody reacts with IATS serotypes 6 and 13 and Fisher immunotype 1. 30. Claims 1 and 12 for human patients susceptible to bacteremia and other diseases caused by Pseudomonas aeruginosa.
, 20, or 25 for the treatment of a host by administering a therapeutically or prophylactically effective amount of the composition. 31, immortal transformed cell threads that secrete human monoclonal antibodies that specifically react with at least two, but fewer than all, IATS serotypes of Pseudomonas aeruginosa. 32, ATCC No. CRL 8941, 9171, or 9
32. The cell line of claim 31 designated as 258. 33. A human monoclonal antibody reactive with an epitope reactive with the monoclonal antibody produced by the cell line of claim 32. 34, at least one human monoclonal antibody, which reacts with at least two, but not all, IATS serotypes of Pseudomonas aeruginosa and covalently linked to said antibody or each of said monoclonal antibodies; A kit for use in detecting the presence of Pseudomonas aeruginosa, comprising a label that provides a detectable signal coupled to a second antibody that is reactive with a second antibody. 35, a human antimicrobial agent that is protective against at least two IATS serotypes of Pseudomonas aeruginosa in combination with an antimicrobial agent, a gamma globulin fraction from human serum immunoglobulin and/or a physiologically acceptable carrier. Consisting of monoclonal antibodies,
A pharmaceutical composition for the treatment or prevention of Pseudomonas aeruginosa infection. 36. Claim 35, wherein the gamma globulin fraction from human serum immunoglobulins is obtained from humans exhibiting high levels of immunoglobulins reactive with the bacterium Pseudomonas aeruginosa and/or its antigenic components. Pharmaceutical composition. 37. The composition according to claim 35, further comprising a monoclonal antibody reactive with Pseudomonas aeruginosa flagella or exotoxin A. 38. Monoclonal antibodies capable of reacting with P. aeruginosa flagella or endotoxin A in humans susceptible to P. aeruginosa infection; at least one additional serotype on lipopolysaccharide molecules of P. aeruginosa a monoclonal antibody capable of reacting with a determinant; a gamma globulin fraction from human serum; a gamma globulin fraction from human serum that exhibits high levels of immunoglobulins reactive with Pseudomonas aeruginosa; or among antimicrobial agents. administering a prophylactic or therapeutic amount of a monoclonal antibody capable of binding to lipopolysaccharide determinants of at least two IATS serotypes of Pseudomonas aeruginosa in combination with one or more prophylactic or therapeutic amounts; An agent for the treatment of humans. 39. A method for determining the presence of Pseudomonas aeruginosa in a sample, characterized in that the sample is combined with a monoclonal antibody reactive with at least two IATS serotypes of Pseudomonas aeruginosa.